Lepage G, Munoz G, Champagne J, Roy C C
Centre de Recherche Pediatrique, Hôpital Ste-Justine et l'Université de Montréal, Quebec, Canada.
Anal Biochem. 1991 Sep 2;197(2):277-83. doi: 10.1016/0003-2697(91)90392-7.
The need for a more specific, reliable, and reproducible technique for the measurement of malondialdehyde (MDA) has prompted modifications of currently available methods based on the formation and recovery of the complex between MDA and thiobarbituric acid (TBA). To 500 microliters of plasma or to 300 mg of liver homogenate, 2 ml of H2O and 500 microliters of 0.5% butylated hydroxytoluene in methanol were added to prevent further formation of MDA. Precipitation of proteins carried out with 200 microliters of 0.66 N H2SO4 and 150 microliters of 10% Na2WO4 (w/v) led to complete recovery of the MDA standard. Maximum formation of the MDA-TBA complex was obtained by adjusting the pH between 2.5 and 4.5 and heating the MDA-TBA mixture at 100 degrees C for 60 min. Extraction of the MDA-TBA complex was a critical step and proved complete with n-butanol at pH less than 0.75. It was then evaporated at 37 degrees C under nitrogen. The MDA-TBA complex solubilized in H2O was shown to be stable for at least 7 days. These preparative steps led to the detection of a single peak that on spectral analysis was identified as pure MDA-TBA. This procedure offers several advantages in terms of specificity, recovery, and reproducibility.
对一种更特异、可靠且可重复的丙二醛（MDA）测量技术的需求，促使人们对基于MDA与硫代巴比妥酸（TBA）形成及回收复合物的现有方法进行改进。向500微升血浆或300毫克肝脏匀浆中加入2毫升水和500微升甲醇中的0.5%丁基化羟基甲苯，以防止MDA进一步生成。用200微升0.66 N硫酸和150微升10%钨酸钠（w/v）进行蛋白质沉淀，可使MDA标准品完全回收。通过将pH调节至2.5至4.5并在100℃加热MDA-TBA混合物60分钟，可使MDA-TBA复合物最大程度地形成。MDA-TBA复合物的萃取是关键步骤，事实证明在pH小于0.75时用正丁醇可实现完全萃取。然后在氮气下于37℃蒸发。溶解于水中的MDA-TBA复合物显示至少7天内稳定。这些制备步骤可检测到一个单峰，经光谱分析鉴定为纯MDA-TBA。该方法在特异性、回收率和可重复性方面具有诸多优势。
